CN102713630A

CN102713630A - Biomarkers for predicting rapid response to HCV treatment

Info

Publication number: CN102713630A
Application number: CN2010800515027A
Authority: CN
Inventors: S-H·邱; Y·朱
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2009-11-14
Filing date: 2010-11-11
Publication date: 2012-10-03
Also published as: NZ599373A; BR112012011221A2; IL219249A0; EP2499493A1; JP2013511030A; KR20120104239A; US20110281747A1; AU2010317996A1; ZA201203420B; RU2012122637A; MX2012005528A; WO2011058084A1; CA2780044A1

Abstract

The present invention relates to biomarkers that are useful for predicting the response of hepatitis C virus infected patients to pharmacological treatment.

Description

Be used to predict biomarker to the rapid answer of HCV treatment

Technical field

The present invention relates to be used to predict the biomarker of replying of the patient of infection with hepatitis C virus to drug therapy.

Background of invention

In the whole world, HCV (HCV) be major health and be chronic liver disease main cause (Boyer, people such as N., J.Hepatol.200032:98-112).The patient who has infected HCV has the risk that worsens to cirrhosis and hepatocellular carcinoma subsequently, and therefore, HCV is the main indications of liver transfer operation.

According to the World Health Organization (WHO), world wide exist to surpass 200,000,000 infected individuals, and infect to 4 million peoples every year at least 3 1,000,000.In case by being infected, about 20% people removes should virus, but all the other people can carry HCV throughout one's life.10% to 20% chronic infection individuality finally develops into cirrhosis disease or the cancer of destroying liver.This virosis is propagated, is propagated, or propagated and vertically be transmitted to the offspring from infection mother or mother carrier with the property mode by the syringe needle that pollutes by blood that pollutes and blood product with the parenteral mode.Because resistance development is rapid, the existing therapy that infects to HCV has the limited clinical benefit, and wherein said existing therapy is limited to recombinant interferon-α separately or the immunization therapy of uniting with the nucleoside analogues 'Libaweilin '.Press for the improved therapeutic agent of effectively defeating chronic HCV infection.

HCV has been categorized as the member of flaviviridae (flaviviridae), wherein said flaviviridae comprises that Flavivirus (flaviviruses), plague virus belong to (pestiviruses) and hepatitis virus belongs to (hepaciviruses), and wherein said hepatitis virus belongs to and comprises HCV (Rice, C.M.; Flaviviridae:The viruses and their replication. edits: Fields, B.N., Knipe in Fields Virology one book; D.M., and Howley, P.M.; Lippincott-Raven publishing house, Philadelphia, Pa.; The 30th chapter, 931-959,1996).HCV is for including the genomic enveloped virus of about 9.4kb sense single stranded rna.Viral genome is made up of the long open read frame (ORF) and 3 ' the short UTR of 5 ' non-translational region (UTR), about 3011 the amino acid whose polyprotein precursors of coding.5 ' UTR is the conservative part of HCV genome topnotch, and very important to the initial sum control of polyprotein translation.

The genetic analysis of HCV identifies divergent 30% the oligogene type that surpasses of six dna sequence dnas.Each genotype contains the hypotype (Simmonds, P.2004J.Gen.Virol. 85:3173-88) of the nucleotide sequence difference of a series of more closely-related 20-25% of demonstrating.Distinguished the hypotype more than 30.In the U.S., about 70% infected individuals has 1a and the 1b type infects.In the Asia, the 1b type is the most general hypotype (X.Forns and J.Bukh, Clinics in Liver Disease 19993:693-716; People such as J.Bukh, Semin.Liv.Dis.199515:41-63).Unfortunately, and 1 type infection ratio 2 types or 3 type genotype infection tolerance treatment more (N.N.Zein, Clin.Microbiol.Rev., 200013:223-235).

Processing is very similar with polyprotein in the gene organization of the non-structural protein part of the ORF of pestivirus and hepatitis virus.These positive chain RNA virus have single big open read frame (ORF), the essential whole virus proteins of coding virus replication.These protein are expressed as polyprotein, and wherein said polyprotein is processed to produce ripe virus protein after the proteinase of leukoprotease and encoding viral is translated altogether and translated.The virus protein that responsible virus genome RNA duplicates approximately is positioned c-terminus.2/3rds of ORF is called non-structure (NS) albumen.For pestivirus and hepatitis virus, ripe non-structure (NS) albumen is made up of p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B from the order successively of aminoterminal to this ORF c-terminus of non-structural protein code area.

The NS albumen of pestivirus and hepatitis virus is total to be the sequence domains of characteristic with the specified protein function.For example, the virus NS albumen in this two papova has the amino acid sequence motif that belongs to the serine protease characteristic and the amino acid sequence motif that belongs to the unwindase characteristic (people such as Gorbalenya, Nature 1988333:22; Bazan and Fletterick, Virology 1989,171:637-639; People such as Gorbalenya, Nucleic Acid Res.1989,17,3889-3897).Similarly, the NS5B albumen of pestivirus and hepatitis virus have the motif that belongs to the RNA polymerase characteristic that RNA instructs (Koonin, E.V. and Dolja, V.V., Crit.Rev.Biochem.Molec.Biol.1993,28:375-430).

The NS albumen of pestivirus and hepatitis virus is directly similarly in the practical function and the function of virus in life cycle.Under both of these case, the NS3 serine protease is responsible for all proteolysis processing (Wiskerchen and Collett, Virology, 1991,184:341-350 of the polyprotein precursor in its downstream, position among the ORF; People such as Bartenschlager, J.Virol.1993,67:3835-3844; People such as Eckart, Biochem.Biophys.Res.Comm.1993,192:399-406; People such as Grakoui, JVirol.1993,67:2832-2843; People such as Grakoui, Proc.Natl.Acad.Sci.USA1993,90:10583-10587; People such as Ilijikata, J.Virol.1993,67:4665-4675; People such as Tome, J.Virol.1993,67:4017-4026).Under both of these case, NS4A albumen is all as co-factor and the effect of NS3 serine protease (people such as Bartenschlager, J.Virol.1994,68:5045-5055; People such as Failla, J.Virol.1994,68:3753-3760; People such as Xu, J Virol.1997,71:5312-5322).The NS3 albumen of this two papova is all also brought into play effect (people such as Kim, Biochem.Biophys.Res.Comm.1995, the 215:160-166 of unwindase; Jin and Peterson, Arch.Biochem.Biophys.1995,323:47-53; Warrener and Collett, J.Virol.199569:1720-1726).At last, the NS5B albumen of pestivirus and hepatitis virus all have the rna polymerase activity that the RNA of prediction instructs (people such as Behrens, EMBO 1996,15:12-22; People such as Lechmann, J.Virol.1997,71:8416-8428; People such as Yuan, Biochem.Biophys.Res.Comm.1997,232:231-235; Hagedorn, PCT WO 97/12033; People such as Zhong, J.Virol.1998,72:9365-9369).

At present, only there is limited quantity can be used for treating the HCV infection through the therapy of approval.Following literature review be used for treating HCV and new and prior treatment method: the R.G.Gish that suppresses HCV NS5B polymerase, Sem.Liver.Dis., 199919:5; Di Besceglie, A.M. and Bacon, B.R., Scientific American, in October, 1999,80-85; G.Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus LiverDisease, Curr.Drug Targ.Infect Dis.20033 (3): 247-253; People such as P.Hoffmann, Recent patents on experimental therapy for hepatitis C virus infection (1999-2002), Exp.Opin.Ther.Patents200313 (11): 1707-1723; People such as F.F.Poordad, Developments in Hepatitis C therapy during 2000-2002, Exp.Opin.Emerging Drugs 20038 (1): 9-25; People such as M.P.Walker, Promising Candidates for the treatment of chronic hepatitis C, Exp.Opin.investing.Drugs 200312 (8): 1269-1280; People such as S.-L.Tan, Hepatitis C Therapeutics:Current Status and Emerging Strategies, Nature Rev.Drug Discov.20021:867-881; People such as R.De Francesco; Approaching a new era for hepatitis C virus therapy:inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase, Antiviral Res.200358:1-16; People such as Q.M.Wang, Hepatitis C virus encoded proteins:targets for antiviral therapy, Drugs of the Future2000 25 (9): 933-8-944; J.A.Wu and Z.Hong, Targeting NS5B-Dependent RNA Polymerase for Anti-HCV Chemotherapy Cur.Drug Targ.-Inf.Dis.20033:207-219.The literature review of having quoted the compound that is in the research and development different phase at present is incorporated herein by reference with its integral body at this.

1a：R＝C(＝O)NH ₂

1b：R＝C(＝NH ⁺)NH ₂

Ribavirin (Ribavirin) (1a; 1-((2R, 3R, 4S, 5R)-3,4-dihydroxy-5-methylol-tetrahydrofuran-2-yl)-1H-[1,2,4] triazole-3-carboxylic acid amide;

) be the antiviral nucleoside analogue of interferon-induced, wide spectrum that synthesize, non-.Ribavirin has the activity of several kinds of DNA of external opposing and RNA viruses, comprising flaviviridae (Gary L.Davis.Gastroenterology 2000118:S104-S114).Though in monotherapy, Ribavirin is reduced to normal level with the serum transamination enzyme level among 40% patient, it can not reduce the serum levels of HCV-RNA.Ribavirin also shows remarkable toxicity, and knownly can induce anaemia.Ribavirin is the suppressant of inosine monophosphate dehydrogenase.Do not ratifying Ribavirin in the monotherapy of HCV, but the conjoint therapy of this compound and Intederon Alpha-2a and Interferon Alpha-2b is through approval.Fixed (Viramidine) 1b of Wella rice is the pro-drug that in liver cell, changes 1a into.

Interferon (IFN) can be used for treating chronic hepatitis 10 years nearly.IFN is the glycoprotein that the immune cell responses virus infections produces.Generally acknowledged interference have two kinds dissimilar: 1 type comprises several kinds of interferon-' alpha 's and a kind of interferon beta, and 2 types comprise interferon gamma.1 type interferon is mainly produced by infected cell, and the protection adjacent cells avoids from the beginning infecting.The IFN inhibition comprises the virus replication of many viruses of HCV, and when it was used for treating hepatitis C infection separately, IFN suppresses serum HCV-RNA extremely can not detected level.In addition, the level of IFN normalization serum aminopherase.Unfortunately, the effect of IFN is a short-term.Stop to treat and to cause 70% recurrence rate, and have only 10-15% to present virology reaction lasting, tool normal serum alanine transaminase level.(L.-B.Davis sees above).

A restriction of early stage IFN therapy is that protein is removed from blood very soon.The chemical derivatization of IFN and polyglycol (PEG) produces the protein of the improved pharmacokinetics character of tool certain degree.

is the conjugate of the mono methoxy PEG of Intederon Alpha-2a and 40KD branch; And

is conjugate (people such as B.A.Luxon, the Clin.Therap.200224 (9): 1363-1383 of Interferon Alpha-2b and 12KD mono methoxy PEG; A.Kozlowski and J.M.Harris, J.Control.Release, 200172:217-224).

At present, the granted conduct of Intederon Alpha-2a and the Interferon Alpha-2b monotherapy that is used to treat HCV.

is the recombinant forms of Intederon Alpha-2a (Roche).

is Pegylation (promptly polyethyleneglycol modified) form of Intederon Alpha-2a (Roche). (Schering Corporation) is the recombinant forms of Interferon Alpha-2b, and

(Schering Corporation) is the Pegylation form of Interferon Alpha-2b.

At present, also researched and developed the interferon-' alpha ' that is used to treat HCV clinical, and other forms of interferon beta, γ, τ and ω.For example,

Ares-Serono of InterMune (interferon alphacon-1), Viragen

(natural interferon), Human Genome Sciences

(interferon beta-1a), the omega interferon of BioMedicine, the oraferon α of Amarillo Biosciences and interferon gamma, interferon-tau and the gamma interferon 1-b of InterMune are developed.

With Ribavirin and interferon-' alpha ' therapeutic alliance HCV is current best therapy to HCV.Associating Ribavirin and Peg (seeing below) produce lasting virus and reply (SVR) in the patient of 54-56%.For 2 types and 3 type HCV, SVR is near 80% (Walker sees above).Unfortunately, conjoint therapy also has side effects, and this gives the clinical challenge that proposed.Depressed, cold like symptoms is relevant with subcutaneous administration IFN-α with dermoreaction, the hemolytic anemia then continued treatment with Ribavirin is relevant.

Now, identified many potential molecule targets that carry out the medicament research and development of anti-HCV therapy, comprising but be not limited to NS2-NS3 oneself protease, N3 proteinase, N3 unwindase and NS5B polymerase.It is the sin qua non that RNA RNA-dependent polymerase duplicates for the rna gene group of strand, justice, and this enzyme has caused medicinal chemist's very big interest.

The nucleosidic inhibitors of NS5B polymerase can be used as the non-natural substrates that causes chain termination or works the competitive inhibitor that combines with polymerase as competition nucleotide.Some NS5B polymerase nucleosidic inhibitors has been disclosed in the following publication, and said whole publications are incorporated herein by reference with it in full.

B=adenine, thymidine, uracil, cytidine, guanine and hypoxanthine

In November 29 calendar year 2001 disclosed WO 0190121, the open and example of J.-P.Sommadossi and P.Lacolla 1 '-alkyl of formula 2 and formula 3-with the anti-HCV polymerase activity of 2 '-alkyl nucleosides.In Dec 6 calendar year 2001 disclosed WO 01/92282, the open and example of J.-P.Sommadossi and P.Lacolla with 1 '-alkyl of formula 2 and formula 3-viral with the plague virus genus with 2 '-alkyl nucleosides treatment Flavivirus.In on April 3rd, 2003 disclosed WO 03/026675, G.Gosselin discloses and has been used to treat 4 '-alkyl nucleosides 4 that Flavivirus and plague virus belong to virus.

In on January 8th, 2004 disclosed WO 2004003000, people such as J.-P.Sommadossi disclose 1 '-, 2 '-, 3 '-with 2 ' of 4 '-substituted β-D and β-L nucleosides-and 3 ' pro-drug.In on January 8th, 2004 disclosed WO 2004/002422, with 2'-C-methyl-3 '-O-L-valine ester ribofuranosyl (ribofuransyl) cytidine is used to treat flaviviridae infections.Idenix has reported the clinical testing of related compound NM283, thinks that this NM283 is the L-valine ester 5 (B=cytimidine) of cytidine analog 2.In on January 8th, 2004 disclosed WO 2004/002999, people such as J.-P.Sommadossi disclose be used to treat comprise 1 ' of flaviviridae infections that HCV infects-, 2 '-, 3 '-or 4 '-branch nucleosides is a series of 2 '-or 3 ' pro-drug.

In on June 3rd, 2004 disclosed WO2004/046331, people such as J.-P.Sommadossi disclose the nucleosides and the flaviviridae sudden change of 2 '-branch.In on April 3rd, 2003 disclosed WO03/026589, the method for the nucleosides treatment HCV that people such as G.Gosselin disclose with 4 '-modified.In on February 3rd, 2005 disclosed WO2005009418, people such as R.Storer disclose and have been used to treat the purine nucleoside analogs that is comprised the disease that HCV causes by flaviviridae.

Other patented claims disclose the purposes of some nucleoside analog treatment infection with hepatitis C virus.In May 10 calendar year 2001 disclosed WO 01/32153, R.Storer discloses the nucleoside derivates that is used to treat virus disease.In August 23 calendar year 2001 disclosed WO 01/60315, people such as H.Ismaili disclose with nucleoside compound treatment or prevention of flavivirus and have belonged to the method that infects.In on March 7th, 2002 disclosed WO 02/18404, people such as R.Devos disclose the 4 '-substituted nucleosides that is used to treat HCV virus.In October 25 calendar year 2001 disclosed WO 01/79246, K.A.Watanabe discloses and has been used to treat 2 ' of virus disease-or 3 '-methylol nucleoside compound.At on the April 25th, 2002 of disclosed WO 02/32920 and on June 20th, 2002 disclosed WO02/48 165, people such as L.Stuyver disclose the nucleoside compound that is used to treat virus disease.

In on Dec 24th, 2003 disclosed WO 03/105770, people such as B.Bhat disclose and have been used to treat a series of carbocyclic nucleoside derivants that HCV infects.In on January 22nd, 2003 disclosed WO 2004/007512, people such as B.Bhat disclose the nucleoside compound that suppresses RNA RNA-dependent varial polymerases.Disclosed nucleosides mainly is 2'-methyl-substituted nucleosides of 2'-hydroxyl in this is open.In on July 25th, 2002 disclosed WO 2002/057425, people such as S.S.Carroll disclose the method that the nucleoside derivates that suppresses RNA dependovirus polymerase and treatment HCV infect.In on July 25th, 2002 disclosed WO02/057287, people such as S.S.Carroll disclose relevant 2 Alpha-Methyls and 2 Beta-methyl ribose derivates, and wherein base is optional substituted 7H-pyrrolo-[2,3-d] pyrimidine radicals 6.Same application discloses an instance of 3 Beta-methyl nucleosides.People such as S.S.Carroll (J.Biol.Chem.2003278 (14): 11979-11984) disclose and utilize 2'-O-methylcytidine (6a) to suppress the HCV polymerase.In on January 29th, 2004 disclosed WO2004/009020, people such as D.B.Olsen disclose a series of sulfo-nucleoside derivates as the suppressant of RNA RNA-dependent varial polymerases.

The title of Emory university discloses with some 2'-fluoro nucleosides treatment HCV for the PCT publication number WO 99/43691 of " 2'-fluoro nucleosides (2'-Fluoronucleosides) ".The title of Emory university is that the U.S. Patent number 6,348,587 of " 2'-fluoro nucleosides " discloses the 2'-fluoro nucleosides family that is used to treat hepatitis B, HCV, HIV and abnormal cell proliferation.Two kinds of configurations of 2' fluorine substituent are also disclosed.

People such as Eldrup (oral lecture V, HCV, flaviviridae; The 16th international conference (Oral Session V, Hepatitis C Virus, Flaviviridae about antiviral research; 16th International Conference on Antiviral Research) (on April 27th, 2003, Savannah, Ga.)) structure-activity relationship that suppresses the 2 ' modified nucleoside of HCV disclosed.

People such as Bhat (oral lecture V, HCV, flaviviridae; About the 16th international conference of antiviral research (on April 27th, 2003, Savannah, Ga.); P A75) synthetic and pharmacokinetics character as the nucleoside analog of the possible suppressant of HCV rna replicon has been described.Author's report, the nucleosides that 2'-modifies show the inhibition activity of strong effect in measuring based on the replicon of cell.

People such as Olsen (oral lecture V, HCV, flaviviridae; About the 16th international conference (on April 27th, 2003, Savannah, Ga.) p A76) of antiviral research the influence of the nucleosides of 2 '-modification to the HCV rna replicon described also.

The non-nucleosides HCV NS5B suppressant of several types has been described; And be incorporated herein by reference in full with it; Comprise benzimidazole (people WO 01/47833 such as H.Hashimoto, people WO 03/000254 such as H.Hashimoto, people WO 03/020240A2 such as P.L.Beaulieu; People US 6,448 such as P.L.Beaulieu, 281B1; People WO 03/007945A1 such as P.L.Beaulieu); Indoles (people WO 03/0010141A2 such as P.L.Beaulieu); Benzothiadiazine, for example, 7, (people WO 01/85172A1 such as D.Dhanak; People WO 03/037262A2 such as D.Dhanak; People WO03/099801A1 such as K.J.Duffy; People WO 2004052312 such as D.Chai; People WO2004052313 such as D.Chai; People WO02/098424 such as D.Chai; People WO2004/041818A1 such as J.K.Pratt; People WO 2004/087577A1 such as J.K.Pratt); Thiophene, for example, 8 (people WO 02/100851A2 such as C.K.Chan);

Benzothiophene (D.C.Young and T.R.Bailey WO 00/18231); Beta-keto pyruvic acid (β-ketopyruvates) (people US 6,492 such as S.Attamura, 423B1, people WO 00/06529 such as A.Attamura); Pyrimidine (people WO 02/06246A1 such as C.Gardelli); Hybar X (T.R.Bailey and D.C.Young WO 00/13708); Triazine (people WO02/079187A1 such as K.-H.Chung); Rhodanine derivant (T.R.Bailey and D.C.Young WO 00/10573, people WO 01/77091A2 such as J.C.Jean); 2,4-dioxo pyrans (people EP256628A2 such as R.A.Love); Phenylalanine derivative (people J.Biol.Chem.2003278:2489-2495 such as M.Wang).

Antiviral (for example, HIV, HCV, simple sore exanthema virus (Herpes simplex), CMV) and anti-cancer chemotherapeutic agents that nucleoside derivates is normally imitated by force.Unfortunately, their practical application receives the restriction of two aspect factors usually.At first, the pharmacokinetics character of difference usually limited nucleosides through the absorption of intestines and limited nucleoside derivates intracellular concentration and, the second, suboptimal physical property has limited the preparation of sending that can be used for the enhanced activity composition and has selected.

Albert introduces the term pro-drug, is used for describing lacking intrinsic BA but energy metabolism changes into the compound (A.Albert, Selective Toxicity, Chapman and Hall, London, 1951) of active medicine.At present, pro-drug (people such as P.Ettmayer, J.Med Chem.200447 (10): 2393-2404 have been carried out summing up; People such as K.Beaumont, Curr.Drug Metab.20034:461-485; H.Bundgaard; The design of pro-drug: the biological transformable derivant of multiple functional group and chemical entities (Design of Prodrugs:Bioreversible derivatives for various functional groups and chemical entities); In Design of Prodrugs one book; H.Bundgaard (editor) Elsevier Science Publishers, Amersterdam 1985; People such as G.M.Pauletti, Adv.Drug Deliv.Rev.199727:235-256; R.J.Jones and N.Bischofberger, people such as Antiviral Res.199527:1-15 and C.R.Wagner, Med.Res.Rev.200020:417-45).Though can pass through specific enzyme, hydrolytic enzyme catalysis metabolic conversion normally also can be through unspecific chemical method regeneration activity compound.

Pharmaceutically acceptable pro-drug refers to metabolism in the host, and for example hydrolysis or oxidation are to form the compound of compound of the present invention.Bio-transformation should avoid forming the fragment with toxicology tendency.The general instance of pro-drug comprises the compound with the unsettled blocking group biologically that is connected with the funtion part of reactive compound.In the design of precursor nucleotide (pronucleotides), used alkylation, acidylate or other lipophilicitys of the oh group on sugar moieties to modify.These precursor nucleotide in vivo hydrolysis or dealkylation to produce reactive compound.

The factor that limits oral bioavailability is normally from GI absorption and the head property the crossed discharge (first-pass excretion) through intestines wall and liver.Optimizedly stride the cell absorption and need D (7.4) greater than 0 through GI.Yet the optimization of partition factor does not guarantee success.Pro-drug must be avoided initiatively discharging transport protein in the enterocyte.Intracellular metabolism can cause passive transportation, perhaps through extraction pump the metabolic product active transport returned the alimentary canal inner chamber in enterocyte.Pro-drug also must be designed to the undesirable bio-transformation of opposing in blood before arriving target cell or acceptor.

Though based on the chemical functional property that exists in the molecule; Sometimes can reasonably design the pro-drug of inferring; But the chemical modification of reactive compound has produced brand-new molecular entity, and it can demonstrate physics, chemistry and the biological property of non-existent non-expectation in parent compound.If a plurality of approach have produced a plurality of metabolic products, require (regulatory requirement) to challenge to the supervision of identifying metabolic product so.Therefore, the evaluation of pro-drug be still undetermined be rich in the practice of challenge.In addition, the pharmacokinetics character of estimating potential pro-drug is challenging and effort costliness.Pharmacokinetics the possibility of result from animal model is difficult to be extrapolated to the mankind.

Recently; Found in the patient who has infected hepatitis c virus genotype 1 (HCV-1) or genotype 4 (HCV-4); If patient's HCV rna level after treatment, be as short as two the week time in become and can not detect, then can predict the useful of treatment that comprises interferon-' alpha ', Ribavirin and HCV AG14361 (triple therapy) replied.The total US patented claim USSN61/138 that submits on Dec 18th, 2008; Disclose in 585 and demonstrate quick virus and reply and obtain to continue virus when the treatment of-2 weeks (RVR2) and triple therapy finishes and reply the association between the patient of (SVR), be incorporated herein by reference with its integral body at this.

Summary of the invention

The present invention is based in the infection of having experienced the triple therapy treatment discovery among the patient of hepatitis c virus genotype 1 (HCV-1) or genotype 4HCV (HCV-4); Suitable biomarker can be predicted the patient who obtains RVR2, and it can be that treatment demonstrates the positive prediction factor that continues the patient that virus replys when finishing subsequently.

In one embodiment, the invention provides and be used to predict that the human experimenter that infected HCV-1 or HCV-4 will obtain the method for RVR2 to the treatment with interferon, Ribavirin and HCV NS5B AG14361, comprising:

(i) before said treatment, the sample from said experimenter (before the treatment) is provided,

(ii) measure at least a protein expression level that is selected from MDC, EC's chemotactic protein (Eotaxin), IL10, TARC and MCPI in the said sample and

(iii) at least a protein expression level and reference value in the said sample are compared, said reference value representes to be derived from least a protein expression level that said treatment is not obtained sample before patient crowd's the treatment of RVR2;

At least a protein in the wherein said sample remarkable higher expression on statistics representes that said experimenter will obtain the RVR2 to said treatment.

In another embodiment, the invention provides and be used to predict that the human experimenter that infected HCV-1 or HCV-4 will obtain the method for RVR2 to the treatment with interferon, Ribavirin and HCV NS5B AG14361, comprising:

(i) after said one week of treatment, the sample from said experimenter (one week of treatment back) is provided,

(ii) measure at least a protein expression level that is selected from TRAIL and IL 12p70 in the said sample and

(iii) at least a protein expression level and reference value in the said sample are compared, said reference value represent to be derived to said treatment do not obtain among the patient crowd of RVR2 one week of treatment the back sample at least a protein expression level;

Still in another embodiment, the invention provides and be used to predict that the human experimenter that infected HCV-1 or HCV-4 will obtain the method for RVR2 to the treatment with interferon, Ribavirin and HCV NS5B AG14361, comprising:

(ii) measure at least a protein expression level that is selected from TGF β 1, MIP1b, TRAIL and MDC in the said sample,

(iii) after said one week of treatment, the sample from said experimenter (one week of treatment back) is provided,

(iv) measure at least a protein expression level that is selected from TGF β 1, MIP1b, TRAIL and MDC in the said sample,

(v) measure from sample before said experimenter's the treatment with from the differential expression level of at least a protein between one week of said experimenter's the treatment back sample; The said differential expression level and the reference value of at least a protein are compared, and said reference value representes to be derived from the differential expression level that said treatment is not obtained at least a protein of sample and one week of treatment back sample before the treatment among the patient crowd of RVR2;

Wherein marked change representes that said experimenter will obtain the RVR2 to said treatment on the statistics of the differential expression level of at least a protein.

The accompanying drawing summary

Fig. 1 has shown the research and design of the II clinical trial phase of RO4588161.

Fig. 2 has shown that 31 group C patients' of the triple therapy treatment of having accepted 1500mg RO4588161,180 μ g Pegasys and Ribavirin RVR2 and SVR treatment reply.

Fig. 3 has shown the protein expression level (in pg/ml) of significant difference (p≤0.05) when showing for 0 week, said significant difference is the significant difference between patient's (representing through " 1 ") who has obtained RVR2 and the patient's (representing through " 0 ") who does not obtain RVR2.

representes mean value, and

expression median.The outlier that is shown as ■ is not included in the mensuration of mean value and median.

Fig. 4 demonstrates the patient's (representing through " 1 ") who obtains RVR2 and does not obtain the protein expression level (in pg/ml) of the significant difference (p≤0.05) between patient's (representing through " 0 ") of RVR2 when showing for 1 week.Symbol have with Fig. 3 in the identical meaning.

Fig. 5 has shown the protein difference expression level (in Δ pg/ml) between 0 week and 1 week, and it has shown the significant difference (p≤0.05) between patient's (representing through " 1 ") who has obtained RVR2 and the patient's (representing through " 0 ") who does not obtain RVR2.Symbol have with Fig. 3 in the identical meaning.

Detailed Description Of The Invention

Term is replying the expectation of one or more medicaments of using to " replying " of treatment.Term can use " continuing virus replys " (" SVR ") and " replying fully " (" CR ") of treatment in this article with exchanging; And refer to when treatment finishes, finish the back during 24 weeks with treatment, not existing in the sample of infected subjects through RT-PCR can detected HCV RNA (＜15IU/mL).Term can use " viral no response " (" VNR ") and " no response " (" NR ") of treatment in this article with exchanging; And refer to during treating and when treatment finishes, existing in the sample of infected subjects through RT-PCR can detected HCV RNA (＞=15IU/mL).Term " fast virus replied for-2 weeks " (" RVR2 ") referred to after two weeks of treatment, not existing in the sample of infected subjects through RT-PCR can detected HCV RNA (15IU/mL).

Term " sample " or " biological sample " refer to from individual isolated tissue or humoral sample; Include but not limited to; For example, cut into slices outward (the external sections of the skin), respiratory tract, enteron aisle and urogenital tract, tear, saliva, breast, haemocyte, tumour, organ of biopsy (tissue biopsy), blood plasma, serum, whole blood, spinal fluid, lymph liquid, skin.Comprise that also cell in vitro cultivates the sample of composition (including but not limited to, by conditioned medium, the virus infected cell of inferring, recombinant cell and the cellular component of the growth gained of cell in nutrient culture media).

Term " reference value of expression expression " refers to the estimation of the average expression of the labelled protein that is derived from HCV patient crowd sample, and said patient crowd demonstrates non-the replying of virus to three treatment therapies.

As looking like at this used term " significant on the statistics " is that the result who is obtained unlikely is because the accidental fluctuation under the particular probability level causes; And it is as used at this; The meaning is to be less than or equal to 0.05 level of significance (p≤0.05), or is less than or equal in 100 5 the probability of error.

The special protein families of species of the height homology that term " interferon " refers to suppress virus replication and cell proliferation and regulate immune response.General suitable interferon includes but not limited to; Interferon Alfa-2b; As from Schering Corporation; Kenilworth; N.J. available A interferon; Interferon Alfa-2a is as from Hoffmann-La Roche, Nutley; N.J. available

interferon; Recombinantinterferon-2C is as from Boehringer Ingelheim Pharmaceutical company limited, Ridgefield; Conn. available

interferon; Interferon alfa-n1 is a kind of purified mixture of natural IFN-, as the Sumitomo from Japan available

or as from the Glaxo-Wellcome company limited of London available

interferon alfa-n1 (INS); Or as at U.S. Patent number 4; 897,471 and 4,695; The total IFN-of described in 623 those (consensus alpha interferon) (particularly their embodiment 7,8 or 9) and from Amgen company limited; Newbury Park, the available specific products of Calif., perhaps make by Interferon Sciences and from Purdue Frederick company; Norwalk; Conn., available Alferon N under the Alferon trade (brand) name is a kind of potpourri of natural IFN-." interferon " can comprise present other forms of interferon-' alpha ' in the clinical development that is used for the HCV treatment, and interferon beta, γ, τ, ω and λ.For example, InerMune

(interferon-' alpha ' con-1), Viragen's

(natural interferon), Human Genome Sciences's (albumin interferon alpha 2 b), Ares-Serono's

The oraferon α of the omega interferon of (interferon beta-1a), BioMedicine, Amarillo Biosciences, and the interferon gamma of InterMune, interferon-tau and gamma interferon 1-b, and Glycoferon ^TM(the total interferon of monoethylene glycol through engineering approaches).Interferon can comprise the interferon like the Pegylation of delimit.

Term " interferon of Pegylation ", " glycol interferon alpha " and " polyglycol interferon " can use with exchanging and refer to interferon-' alpha ', the preferably polyethyleneglycol modified conjugate of Intederon Alpha-2a and α-2b in this article.Generally suitable pegylated interferon α include, but are not limited to and

other forms of pegylated interferon may include ZymoGenetics and Bristol-Myers? Squibb of PEG-interferon λ.

Term " Ribavirin " refers to compound 1-((2R; 3R; 4S; 5R)-3; 4-dihydroxy-5-methylol-tetrahydrofuran-2-yl)-1H-[1; 2,4] triazole-3-carboxylic acid amide, it is that antiviral nucleoside analogue interferon-induced, wide spectrum that synthesize, non-also can obtain under title

and .

Term " RO4588161 " is used in reference to compound isobutyric acid (2R, 3S, 4R in this article; 5R)-and 5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-2-azido-3,4-two-isobutyl acyloxy-tetrahydrofuran-2-base methyl esters comprises the pharmaceutically acceptable acid addition salts; And with people such as P.J.Pockros, Hepatology, 2008; Disclosed term " R1626 " uses interchangeably among the 48:385-397, and said document is incorporated herein by reference with it in full.

Term " RO5024048 " is used in reference to compound in this article, isobutyric acid (2R, 3R; 4R, 5R)-5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-4-fluoro-3-isobutyl acyloxy-4-methyl-tetrahydrofuran-2-base methyl esters, comprise the pharmaceutically acceptable acid addition salts; And with people such as S.Ali; Antimicrob Agents Chemother., 200852 (12): disclosed term " R7128 " uses interchangeably among the 4356-4369, and said document is incorporated herein by reference with it in full.

Term " about 2 weeks " refers to two weeks or 1-2 days the time period of adding deduct in 14 days.

Term " MDC " refers to the chemotactic factor (CF) that macrophage is derived, and it also is called chemotactic factor (CF) (C-C motif) part 22 or CCL22, and its human protein sequence is disclosed in GenBank accession number NP_002981.

Term " EC's chemotactic protein " refers to the eosinophil chemotactic protein; It is also referred to as EC's chemotactic protein-1 and chemotactic factor (CF) (C-C motif) ligand 11 or CCL11, and its human protein sequence is disclosed in GenBank accession number NP_002977.

Term " IL10 " or " IL-10 " refer to interleukin-11 0, and it is also referred to as IL 10A or CSIF, and its human protein sequence is disclosed in GenBank accession number NP_000563.

Term " TARC " refers to thymus gland and activates the chemotactic factor (CF) of regulating, and it is also referred to as chemotactic factor (CF) (C-C motif) ligand 17 or CCL17, and its human protein sequence is disclosed in GenBank accession number NP_002978.

Term " MCP1 " refers to the albumen 1 of monocyte chemoattractant albumen 1 or monocyte chemotaxis, and it is also referred to as chemotactic factor (CF) (C-C motif) part 2 or CCL2, and its human protein sequence is disclosed in GenBank accession number NP_002973.

Term " TRAIL " refers to the relevant apoptosis induction ligand of TNF-, and it is also referred to as TNF (part) superfamily, member 10 or TNFSF10, and Apo-2L, and its human protein sequence is disclosed in GenBank accession number NP_003801.

Term " IL 12p70 " or " IL-12p 70 " refer to the biologically active form of interleukin 12 (IL12/IL-12); Heterodimer by the disulfide bonding between IL 12p35 and the IL 12p40 is formed; IL 12p35 is also referred to as interleukin 12 A or IL 12A; And its human protein sequence is disclosed in GenBank accession number NP_000873, and IL 12p40 is also referred to as interleukin 12 B or IL12B, and its human protein sequence is disclosed in GenBank accession number NP_002178.

Term " TFG β 1 ", " TGFbeta1 " refer to transforminggrowthfactor-(β 1), and its human protein sequence is disclosed in GenBank accession number NP_000651.

Term " MIP1b " or " MIP-1b " refer to macrophage inflammatory protein 1-β; It is also referred to as chemotactic factor (CF) (C-C motif) part 4 or CCL4 and lymphocyte-activated gene 1, and its human protein sequence is disclosed in GenBank accession number NP_002975.

For the patient who suffers from chronic hepatitis C, the first-line treatment of recommending at present is 48 weeks of patient of carrying genotype 1 or 4 viruses with pegylated interferon alfa associating ribavirin therapy, and 24 weeks of patient of genotype 2 or 3 viruses are carried in treatment.Discovery is in the patient that one or more interferon-' alpha ' recurred after the course of treatment, and is and among the former patient who did not treat, more effective than interferon-' alpha ' monotherapy with the Ribavirin therapeutic alliance.Yet Ribavirin shows pronounced side effects, comprises teratogenesis and carcinogenicity.In addition, Ribavirin can cause hemolytic anemia in about patient of 10% to 20%, and this needs dosage to reduce or ends ribavirin therapy, and said hemolytic anemia maybe be relevant with the accumulation of Ribavirin triphosphate in red blood cell.Therefore, in order to reduce the generation of medical expense and rough sledding, expectation be under the situation that does not influence effect, treatment to be adjusted into the short duration.

Many researchs show that the quick virus during 4 weeks is replied (RVR) has become the relative reliable prediction (predictor) that the lasting virus of using polyglycol interferon/ribavirin therapy is replied (SVR).Some researchs show, in the HCV-1 patient who has realized RVR, it is suitable (people such as D.M.Jensen, Hepatology, 2006,43:954-960 that SVR leads between 24 weeks and 48 all polyglycol interferon/ribavirin therapy; People such as S.Zeuzen, J.Hepatol.2006,44:97-103; People such as A.Mangia, Hepatology, 2008; 47:43-50), and other researchs show, even reached RVR; In HCV-1 patient, the polyglycol interferon/ribavirin therapy in 24 weeks is still than the treatment in 48 weeks poor (people such as M.-L.Yu, Hepatology; 2008,47:1884-1893).

Embodiment

Embodiment 1: the II clinical trial phase that relates to RO4588161

This be the 2A phase, polycentric, at random, double blinding (RO4588161 and Ribavirin be double blinding and Pegasys is (the open labeled) that openly indicates), test active control, that have ongoing parallel-group research.During 35 days screening (from the first time screening and assessment to the time of for the first time using testing drug) the treatment part (Fig. 1) of having carried out test.During screening, confirm each patient's HCV genotype and HCV RNA titre, and only registered HCV genotype-1 and HCV RNA titre >=50, the not treatment patient of 000IU/mL.

Having registered the masculinity and femininity patient of 107 ages between 18 years old to 66 years old studies.The patient is divided into 4 treatment groups at random:

Group A/Dual 1500 [every day twice, oral RO45881611500mg+ is weekly, subcutaneous Pegasys 180 μ g] continues 4 week-21 patient,

Group B/Dual 3000 [every day twice, oral RO45881613000mg+ is weekly, subcutaneous Pegasys 180 μ g] continues 4 week-34 patient,

Group C/Triple 1500 [every day twice, oral RO45881611500mg+ is weekly, 180 μ g+ every days of subcutaneous Pegasys oral Ribavirin 1000mg (＜75kg) or 1200mg (>=75kg)] continue 4 week-31 patient or

Group D/ standard care (SOC) [weekly, 180 μ g+ every days of subcutaneous Pegasys oral Ribavirin 1000mg (＜75kg) or 1200mg (>=75kg)] continue 4 week-21 patient

, do not accept the research medicine of single dose, so in 107 patients altogether, there are 104 patient's data can be used for assay because though 3 patients are at random.In 104 patients, because security reason, always have 43,4 and 5 patients respectively and cancelled RO4588161, Pegasys and ribavirin therapy in advance.

The patient that will meet all criterion of acceptability accepts to be with or without around Ribavirin and the RO4588161 Pegasys associating at random or accepts standard care (SOC).

All patients that accepted at least one Research on dose medicine continue to accept Pegasys 180 μ g that weekly subcutaneous disclosing indicate and oral once a day Ribavirin 1000mg (＜75kg) or 1200mg (>=75kg) to accomplish for 48 all total treatment phases.

Through the ratio of PK subgroup group (sparse pharmacokinetics (sparse PK) is to intensive pharmacokinetics (intensive PK)) with 2:3:3:2; Grab sample (randomization) is divided into following treatment group (group A/Dual 1500 ~ 20; Group B/Dual 3000 ~ 30; Group C/Triple 1500 ~ 30, group D/SOC ~ 20).

In the 8th week, promptly use 4 weeks behind the experiment drug regimen of last potion, all patients have been carried out security followed up a case by regular visits to.During with the standard care therapy, the patient is carried out this 4 all security follow up a case by regular visits to.After treatment is accomplished, follow the tracks of 24 weeks of patient of having accomplished whole 48 weeks treatments.

The pharmacokinetics analysis comprises assessment serum-virus carrying capacity, and the virus when the clinical prescription on individual diagnosis of individuality is visited is replied and is evaluated among the patient who did not treat who treats trouble chronic hcv genotype 1 virus infections with the antiviral resistance that is with or without RO4588161 generation Ribavirin and the Pegasys associating.With virus reply be defined as have with Roche COBAS TaqMan HCV thermometrically can not detected HCV RNA (＜15IU/mL) patient's percentage.Pharmacokinetic data be expressed as tabulation, sum up statistics (comprise mean value, median, standard error, mean value fiducial interval, scope, the coefficient of variation, have the patient's who replys the ratio and the fiducial interval of ratio) and mean value figure in time.

In order to identify the protein biomarker of replying for predictability to various therapeutic schemes; Plasma sample is collected from every patient during one week (1 week of time point) in (0 week of time point) and treatment back before treatment, and uses available from Aushon Biosystems (Billerica; MA) special-purpose SearchLight 55-multichannel is sandwich-the ELISA system; Through Moody, M.D. etc., " Assay-Based ELISAs for High-Throughput Analysis of Human Cytokines (being used for the test based on ELISA of human cell factor high throughput analysis) "; Biotechniques; 2001,31 (1): the experimental program described in the 186-194 (it all being incorporated herein by reference) at this, test the expression of various cell factors and chemotactic factor (CF).The human cell factor and the chemotactic factor (CF) of test in the test of 55-multichannel have been listed in the table 1.

Table 1

After with RO4588161, Pegasys and ribavirin therapy, the dosage of observing virus load in the blood plasma relies on and reduces and the time-dependent minimizing.The decline of HCV RNA has just been observed in (72 hours) during assessment first behind first time dosage.When the 4th week, average HCV RNA (IU/mL) has>=3.6log all groups that contain RO4588161 with respect to baseline ₁₀Reduction, all greater than 2.4log with SOC ₁₀

Dual 1500 and Dual 3000 demonstrate dose-dependent minimizing, but have negative 0.9log ₁₀The mean variation difference of the virus concentration of IU/mL (3.6 couples-4.5).When Dual 1500 relatively and Triple 1500 (RO4588161 of same dose and Pegasys, but Ribavirin is arranged), difference even bigger is negative 1.6log ₁₀IU/mL (5.2 couples-3.6).In addition, when SOC relatively and Triple 1500 (Pegasys of same dose and Ribavirin, but RO4588161 is arranged), difference is the most remarkable, is to bear 2.8log ₁₀IU/mL (5.2 couples-2.4).In addition, 95% fiducial interval between Triple 1500 and the Dual1500 and between Triple 1500 and the SOC is not overlapping, shows that the antiviral effect of Triple1500 is more excellent than Dual 1500 and SOC.

31 treatment results of organizing C patients that presented in diagrammatic form among Fig. 2 and experienced triple therapy.2 whens week of treatment (, RVR2) can demonstrate among 13 patients of undetectable HCV RNA, 11 can obtain SVR accomplishing the treatment back during 24 weeks.On the contrary, do not appear among 18 patients of RVR2, have only seven to obtain SVR.

Use Wilcoxon to be lost and check (nonparametric method), will from 55 kinds of chemotactic factor (CF)s and cell factor in the plasma sample before the patient's who has obtained RVR2 the treatment each expression and compare from these protein expression levels in the plasma sample before the patient's who does not obtain RVR2 the treatment.Similarly, will compare from the protein expression level in the sample after RVR2 patient's 1 week of treatment and from the protein expression level in the sample after non-RVR2 patient's 1 week of treatment.In addition, in the differential expression level (δ) of checking and compare every kind of protein between 0 all samples and the 1 all samples between RVR2 patient and the non-RVR2 patient.Critical level 0.05 has been considered statistically-significant difference.Program Spotfire (Spotfire DecisionSite version 9.1.1,2008, TIBCO, Somerville analyzes in MA).Shown in the table 2 when 0 week, 1 week and all difference in 0 week-1 (δ), between RVR2 and non--RVR2, demonstrated the protein of the statistically-significant difference of expression.The expression data of each of these protein of three test points are illustrated among Fig. 3,4 and 5.

Table 2

Claims

1. be used for predicting that the people experimenter who has infected hepatitis c virus genotype 1 (HCV-1) or hepatitis c virus genotype 4 (HCV-4) to obtaining the method that quick virus replied for-2 weeks (RVR2) with interferon, Ribavirin and the treatment of HCV NS5B AG14361, comprising:

(ii) measure at least a protein expression level that is selected from MDC, EC's chemotactic protein, IL10, TARC and MCP1 in the said sample and

2. the process of claim 1 wherein at least two kinds of protein expression levels of having measured.

3. claim 1 or 2 method have wherein been measured at least three kinds of protein expression levels.

4. be used for predicting that the people experimenter who has infected hepatitis c virus genotype 1 (HCV-1) or hepatitis c virus genotype 4 (HCV-4) to obtaining the method that quick virus replied for-2 weeks (RVR2) with interferon, Ribavirin and the treatment of HCV NS5B AG14361, comprising:

5. the method for claim 4 has wherein been measured at least two kinds of protein expression levels.

6. be used for predicting that the people experimenter who has infected hepatitis c virus genotype 1 (HCV-1) or hepatitis c virus genotype 4 (HCV-4) to obtaining the method that quick virus replied for-2 weeks (RVR2) with interferon, Ribavirin and the treatment of HCV NS5B AG14361, comprising:

(iii) after said one week of treatment, the sample from said experimenter (one week of treatment back) is provided, and measures at least a protein expression level that is selected from TGF β 1, MIP1b, TRAIL and MDC in the said sample,

(iv) measure from sample before said experimenter's the treatment and from the differential expression level of at least a protein between one week of said experimenter's the treatment back sample and

(v) said differential expression level and the reference value with at least a protein compares, and said reference value representes to be derived from the differential expression level that said treatment is not obtained at least a protein of sample and one week of treatment back sample before the treatment among the patient crowd of RVR2;

7. the method for claim 6 has wherein been measured the differential expression level of at least two kinds of protein.

8. claim 6 or 7 method have wherein been measured the differential expression level of at least three kinds of protein.